Journal of Microbiology Research

p-ISSN: 2166-5885    e-ISSN: 2166-5931

2022;  12(1): 13-23

doi:10.5923/j.microbiology.20221201.02

Received: Jan. 30, 2022; Accepted: Feb. 11, 2022; Published: Feb. 24, 2022

 

Incidence of Antibiotic Resistant Staphylococcus aureus in Leafy Greens and Clinical Sources

Cherinet Yigrem1, Broderick Eribo2, Kotu Sangre2

1Department of Biology, Howard University, 400 Bryan St NW Washington DC, USA

2Department of Biology, Howard University, 415 College St. NW Washington DC, USA

Correspondence to: Cherinet Yigrem, Department of Biology, Howard University, 400 Bryan St NW Washington DC, USA.

Email:

Copyright © 2022 The Author(s). Published by Scientific & Academic Publishing.

This work is licensed under the Creative Commons Attribution International License (CC BY).
http://creativecommons.org/licenses/by/4.0/

Abstract

Staphylococcus aureus is a nosocomial pathogen associated with serious community and healthcare facility acquired infections. The current study determined the occurrence and diversity of antibiotic resistance Staphylococcus aureus in leafy vegetables sold in Gondar retail markets and clinical sources from Gondar community hospital. The microbiological analyses were performed based on standard methods. The vegetable samples were plated onto plate count agar and incubated for 48h at 30°C. The aerobic plate count of the vegetable samples ranged from 5.3 to 8.3 log10 cfu/g. Among the retail vegetable samples, 31 (28.18%) of them were positive for S. aureus and the average bacterial count was 8.85 cfu/g. The prevalence of S. aureus was highest in cauliflower (13/15, 86.7%), lettuce (13/15, 86.7%), followed by spinach (7/15, 46.7%), and cabbage (2/15, 13.4%). The isolates were analysed for antibiotics susceptibility test, and most isolates (99.3%) were resistant to vancomycin, ampicillin, penicillin, erythromycin, and methicillin. All the vegetable and clinical isolates (61, 100%) were multidrug resistant. There were no significant differences in phenotypic antibiograms between vegetable and clinical isolates (P=0.097>0.05). Phylogenetic tree determined the relationship between the vegetable isolates and the clinical isolates. Macrolide and beta lactam resistant genes and virulence factors were determined among the bacterial population. Four resistant genes were analysed in ten of the antibiotic resistant isolates using colony PCR and the results revealed that 90% were encoding (erm A), 100% (ermB), 100% (erm C), and 70% (msrA) resistant determinants. The present study also determined Hla, sea, Hlb, tst, and eta virulence factors and plasmids of the bacterial isolates.

Keywords: Antibiotic resistant, Resistance genes, Staphylococcus aureus, Plasmid, Virulence factors

Cite this paper: Cherinet Yigrem, Broderick Eribo, Kotu Sangre, Incidence of Antibiotic Resistant Staphylococcus aureus in Leafy Greens and Clinical Sources, Journal of Microbiology Research, Vol. 12 No. 1, 2022, pp. 13-23. doi: 10.5923/j.microbiology.20221201.02.

Article Outline

1. Introduction
2. Materials and Methods
    2.1. Sample Collection
    2.2. Antimicrobial Susceptibility
    2.3. Plasmid Profile and 16s rRNA Sequencing of Bacteria Isolates
    2.4. Polymerase Chain Reaction (PCR) Amplification
    2.5. Primers and PCR
    2.6. Statistical Analysis
3. Result
    3.1. Aerobic Mesophilic Count
    3.2. Antibiotic Susceptibility
    3.3. Analysis of 16S rRNA gene Sequencing
    3.4. Plasmid Profiling of S. aureus Isolates
    3.5. Analysis of Virulence Genes in Staphylococcus aureus Isolates
    3.6. Analysis of the Macrolide and MRSA Resistance Gene Regions
4. Discussions
5. Conclusions
ACKNOWLEDGEMENTS
Conflict of Interest

1. Introduction

Staphylococcus aureus became one among the foremost common causes of nosocomial infections. Multiple antibiotic resistant Staphylococcus aureus pose a growing problem for human health globally [20,29]. The increase of antibiotic resistant virulent strains of Staphylococcus aureus, particularly methicillin resistant S. aureus (MRSA), may be a significant issue within the treatment and control of staphylococcal infections [61,37].
Although, S. aureus is susceptible to known antibiotics [46,32,5], however, there are some antibiotic resistant strains [53,32]. Antibiotic resistance genes expressed by these strains are mainly acquired from external sources. This might be either natural or due to human actions mainly by antibiotic abuse, misuse and cause mutation and antibiotic selection [15,44]. The emergence and global dissemination of antibiotic resistant strains of S. aureus like methicillin resistant S. aureus (MRSA) is of health and socio-economic importance. Hence, antibiotic resistant strains are a major concern globally [26,44]. Annually, about 23, 000 people die due to antibiotic-resistant bacterial infections. Drug-resistant infections or “superbugs” could also cost a cumulative $100 trillion of economic output by 2050 if the world does not act to slow down the rise of drug resistance [7,38,34].
One of the difficulties for therapeutic treatment of S. aureus infection is resistance to several commonly used antibiotics [7,38]. When S. aureus was first discovered, it had been easy to treat using available antibiotics. Meanwhile, after the discovery of penicillin sixty years ago to combat S. aureus, there have been strains of the pathogen that were immune to these antibiotics. A novel therapeutic agent called methicillin was developed and introduced to treat penicillin resistant S. aureus strains in 1961 [33]. The antibiotics, vancomycin, penicillin, and methicillin mode of action is extremely similar, and it involves inhibiting the synthesis of cell membrane through the stoppage of peptidoglycan formation by the pathogen and eventually lysis of the bacterium. However, a year after the introduction of methicillin, strains of S. aureus were reported to be methicillin resistant and gradually these strains disseminated worldwide. MRSA became a deadlier strain, which has become immune to most β- lactam antibiotics [19,56]. The antibiotic resistance of S. aureus is usually considered related to specific resistance genes. Resistance to macrolides (erythromycin and clindamycin) in staphylococci was supported by the active efflux mechanism encoded by the msrA gene, on the other hand resistant to β-lactams was supported by modification on 23S rRNA methylation encoded by ermA, ermB, and ermC genes [42,22]. Additionally, resistance of S. aureus to tetracycline was associated with tetM and tetK genes [48].
Staphylococcus aureus also, harbors genes encoding a variety of virulence factors including enterotoxins (SEs; sea to see, seg to sei, ser to set), exfoliative toxins (eta, etb), toxic shock syndrome toxin-1 (tst), Panton-Valentine leukocidin (lukS/F-PV), staphylococcal complement inhibitor (scn), and hemolysins (hly/hla, hlb, hld, hlgA, hlgB, hlgC) [72,73]. Enterotoxin production is important as it causes food poisoning, whereas the other virulence factors are associated with infection rather than intoxication. The scn gene is a marker of the immune evasion cluster (IEC) of strains that has been detected at high frequency among diverse collections of S. aureus strains obtained from humans, indicating that the scn gene may be useful for differentiating strains that have been transmitted from livestock to humans from those of human origin [74].
Although there were handful of studies on antibiotic resistance S. aureus pathogens in Gondar Ethiopia, most of the articles focused on those pathogens isolated from meat, poultry, and milk in contrast, little information was found on vegetables [41]. Additionally, most of the works done regarding the incidence of antibiotic resistant bacteria are phenotypic, there are few molecular studies made in determining the resistant genes and virulence factors [21]. Therefore, the current study addressed the incidence and distribution of antibiotic resistant Staphylococcus aureus in leafy vegetables sold in Gondar retail markets related to clinical isolates from Gondar University Hospital. The article further determined whether leafy vegetables play a role as a carrier and reservoir of antibiotic resistant genes and identified virulence factors and plasmids among the bacteria population.

2. Materials and Methods

2.1. Sample Collection

A total of 112 retail vegetable samples including cauliflower, spinach, lettuce, and cabbage, were collected from three different retail markets in Gondar, Ethiopia. The vegetable samples were placed in a cold box at a temperature approximately 4°C, tightly sealed with sterile plastic wrap, and transported to an accredited laboratory and subjected to microbiological analysis within 24 hrs. Among the total vegetable samples 31 of the isolates were confirmed as S. aureus. A total of 30 clinical strains were collected from Gondar university hospital. The isolates were identified from different specimens and no duplicate isolates from the same patient were included in this study. The organisms were identified to the species level using standard biochemical methods. S. aureus was identified by Gram staining, production of coagulase, catalase, DNase, and oxidation and fermentation of mannitol.
The aerobic mesophilic count of S. aureus isolates in the vegetable samples were determined by the standard plate count method as described by the American Public Health association (APHA) on plate count agar [11]. Four tenfold serial decimal dilutions were made for each sample; 1 mL of each step was inoculated into duplicates of plate count agar (Himedia). For aerobic mesophilic plate count, the plates were incubated at 37°C for 24 h, and for the psychotropic plate count, incubated at 6°C for 5–7 days. The colonies formed on the plates were counted and expressed as log10 colony forming unit/g (log10 cfu/g) [43].

2.2. Antimicrobial Susceptibility

To detect the S. aureus isolates for antibiotic susceptibility, the Kirby Bauer method [24] was performed by standard disk diffusion on Mueller–Hinton agar and incubated at 37°C for 24 h, following the guidelines of the Clinical and Laboratory Standards Institute [10]. A total of 25 antibiotics (Oxoid, Basingstoke, UK) were classified into 14 different groups [56]: amoxycillin/clavulanic acid (AMC, 30μg), ampicillin (AMP, 10 μg), cefepime (FEP, 10 μg), Cefoxitin (FOX, 30 μg), penicillin G (P, 10U), ceftazidime (CAZ, 30 μg), amikacin (AK, 30 μg), gentamicin (CN, 10 μg), kanamycin (K, 30 μg), streptomycin (S, 25 μg), chloramphenicol (C, 30μg), clindamycin (DA, 2μg), erythromycin (E, 15 μg), telithromycin (TEL, 15 μg), ciprofloxacin (CIP, 5 μg), Norfloxacin (NOR, 10μg), tetracycline (TE, 30μg), Linezolid (LZD, 30μg), rifampicin (RD, 5μg), Trimethoprim/sulphamethoxazole (SXT, 25 μg), Quinupristin/dalfopristin (QD, 15 μg), Teicoplanin(TEC, 30μg), Methicillin( 10μg), Nitrofurantoin (F, 300 μg) and Vancomycin (Van, 10 μg). Staphylococcus aureus ATCC 29213 and Escherichia coli ATCC25922 were included for quality control [11].

2.3. Plasmid Profile and 16s rRNA Sequencing of Bacteria Isolates

The isolates were subjected to plasmid profile analysis via the OMEGA miniprep method. An overnight culture of every bacteria isolate was prepared in 5 mL of nutrient broth [44]. The broth culture was properly mixed by vertexing, and 1.5 mL was then transferred into a prelabeled Eppendorf tube. The tubes were then centrifuged for two minutes at 13 000 revolutions per minute (rpm) to reap the bacterial cells. The supernatant was gently decanted, leaving about 100 μL of the broth culture, which was then vortexed at high speed until the bacterial cell pellet became completely suspended [45]. OMEGA solution (300 μL; 25mM Tris, 10mM EDTA, 0.1N NaOH, 0.5% SDS) was then added to lyse the bacterial cells. The answer was mixed by inversion 3 for five times until the answer became slimy, after which 150 μL of three .0M sodium acetate (PH 5.2) was added and again vortexed for about 10 seconds [10]. The answer was centrifuged at 13 000 rpm for five minutes to discard cellular debris and chromosomal DNA [44]. The supernatant was then transferred into another labeled 1.5-mL Eppendorf tube, and 900 μL of cold absolute ethanol was added. The answer was centrifuged at 13 000 rpm for 10 minutes. The supernatant was discarded, and therefore the white pellet containing the plasmid DNA was rinsed twice with 1000 μL of 70% ethanol [26].
The pellet was then air-dried, and 40 μL of TE buffer (10mM Tris, 1mM Na2EDTA) was added to the resuspend the pellet [45]. Electrophoresis of the extracted plasmid DNA was administered on 1.0% agarose gel and visualized with ethidium bromide via the ultraviolet transilluminator [45]. Selected and representative isolates were sent to GENWIZ for sequencing of the 16S rRNA genes.

2.4. Polymerase Chain Reaction (PCR) Amplification

Colony PCR was performed to detect all selected genes using the protocols listed in Table 1. The PCR mixture was obtained from EconoTaq PLUS and EconoTaq PLUS GREEN 2X Master Mixes, and each reaction was conducted under proper conditions: initial denaturation at 94°C for 4 minutes, followed by 35 cycles of 94°C for 45 seconds, polymerization at 72°C for 1 minute, and final extension for five minutes and 1 minute of annealing. Amplified products of all virulence factors are going to be further analyzed using ethidium bromide-mixed agarose gel (1.8%) under a UV Tran illuminator. Resistant genes were determined from the isolate supporting protocols listed in Table 1 and using standard primers (table 2) by performing colony PCR. Additionally, PCR was used to detect the presence of virulence genes: hla, hlb, sea, seb, sec, pvl, eta, etb, and tsst [34,22] mecA gene amplification was performed and identify MRSA isolates. SCCmec typing of MRSA strains was analyzed by multiplex PCR as previously described based on the primer’s conditions listed in table 3 [18].
Table 1. Reaction set up for all resistance genes and virulence factors
     

2.5. Primers and PCR

The four primer pairs used to amplify the ermA, ermB, ermC, and msrA genes are listed in table 2 [51]. Within the current study, colony polymerase chain reaction (PCR) amplification was performed for ermA, ermC, and msrA genes as previously described [62] during a 25 µL reaction mixture consisting of: 1X PCR reaction buffer, 2.5 mM MgCl2, 0.2 mM dNTP, 25 per mol of every of the three primer pairs, 1.2 U of Taq DNA polymerase (Fermentas, Lithuania) and 200 ng DNA template. For ermB gene amplification, the ermB primers (25 per mol) were added during a separate PCR reaction under an equivalent above-mentioned condition. Amplification was administered employing a gradient thermal cycler (Bio-Rad, my cycler, US) because the following program: five minutes at 94°C, 30 cycles of amplification, consisting one minute at 95°C, 0.5 minutes at 55°C and two minutes at 72°C, ending with 10 minutes at 72°C (final extension). To verify amplification, 5 μL of PCR products were subjected to agarose gel electrophoresis (1.5% agarose, 1X TAE, 100V). Ethidium bromide-stained DNA fragments were then visualized with a UV transilluminator at 300 nm as compared to a 50bp/100bp molecular size standard ladder [21,16,13]. Additionally, PCR was used to detect the presence of nine virulence genes: hla, hlb, sea, seb, sec, pvl, eta, etb, and tsst [2,27,53]. All primers and the PCR amplification condition of resistance genes and virulence factors were listed in tables 2 and 3.
Table 2. List of primers used for the detection of genes encoding antibiotic resistance
     
Table 3. Primers used for PCR of virulence factor detection

2.6. Statistical Analysis

To determine the aerobic mesophilic count among the vegetable samples and vegetable markets, serial dilution and log10 cfu/gm of colony counting time dilution factor divided by total volume were used. Differences in overall vegetable bacterial distribution among the vegetable types cauliflower, lettuce, spinach and cabbage were analyzed using average and percentage. Positive correlations in the antibiotic sensitivity tests between the bacterial isolates of the vegetable samples and clinical sources were assessed using one-way ANOVA. The z- tests were used to analyze the difference in resistance genes among the bacterial isolates. Significant differences in bacterial populations were observed across vegetable types using the nonparametric Kruskal-Willi’s test and between environmental samples and clinical sources employing a t-test.

3. Result

3.1. Aerobic Mesophilic Count

The vegetable types, number of samples, counts of standard deviation (SD), and average (mean) in cfu/g of aerobic colony count are provided in table 4 below. The total bacterial count result indicated that all vegetable samples contain high bacteria load. The mean aerobic mesophilic count of the leafy vegetables was (7.9 cfu/g) cauliflower, (6.9 cfu/g) lettuce, (6.7 cfu/g) spinach and (6.5cfu/g) cabbage.
Table 4. Aerobic mesophilic count of vegetable samples
     

3.2. Antibiotic Susceptibility

The antimicrobial susceptibility patterns of Staphylococci isolated to various antibiotics are presented in table 3. Out of the total 31 vegetable isolates, 30(95.2%) exhibited resistance to penicillin, 31(100%) resistance to methicillin, and 31(100%) resistance to erythromycin. Most of these isolates were also resistant to tetracycline, levofloxacin, vancomycin, and gentamicin. Among the 30 clinical isolates of Staphylococci spp. 29(97%) were resistance to penicillin, 30(100%) resistance to erythromycin, and 30(100%) resistance to methicillin. Overall, each of the isolates showed multiple antibiotic resistant (resistant to more than three classes of antibiotics) in both vegetable and clinical isolates. Comparing the antibiogram of both sample sources shows similarity, P=0.097>0.05 (table 5 and figure 1).
Table 5. Antibiotics susceptibility results for clinical and vegetable isolates
     
Figure 1. Antibiotic susceptibility pattern of vegetable and clinical Staphylococcus aureus isolates (the first three R, I, S resistance pattern vegetable isolates and the second three R, I, S showed clinical isolates) R= resistance, I= intermediate, S=sensitive

3.3. Analysis of 16S rRNA gene Sequencing

The sequences obtained in this study showed similarities between the sequences that obtained from clinical sources and sequences obtained from the vegetable samples respectively (MN872295, MN871695, MN871721, MN871820, MN872300, MN872311) clinical sources (MN871892, MN871924, MN871978, MN871721, and MN871988) vegetable samples as indicated in Fig. 2.
The tree was constructed using the nucleotide sequences of eight 16s rRNA genes isolated from staphylococci isolates from clinical source and vegetable samples and one sequence obtained from the Gen bank as mentioned before (NR-118997.2). All sequences were clipped to an identical length corresponding to the same region before creating the tree (Fig. 2). The tree showed a high degree of similarity between all sequences of vegetable and clinical isolates or those imported from the Gen bank. The sequence of isolates (MN872295, MN871695, MN871721, MN871820, MN872300, and MN872311) that obtained from clinical sources, and sequences (MN871892, MN871924, MN871978, MN871721, and MN871988) obtained from vegetable samples respectively, are closer to each other and formed closed lineage (Figure 2).
Figure 2. Evolutionary relationship of 16s rRNA sequenced Staphylococci isolates (NR-118997.2 S. aureus strain ATCC12600 from NCBI)

3.4. Plasmid Profiling of S. aureus Isolates

Plasmid was isolated from both leafy vegetable isolates and clinical sources. Out of the twenty-one vegetable isolates, plasmids were detected in 13(61.9%) of the S. aureus isolates (Figure. 3A). The clinical isolates 10(100%) were found to have at least one plasmid (Figure 3. A1 & A2).
Figure 3. Plasmid profile of Staphylococcus aureus A1) Vegetable and A2) Clinical isolates

3.5. Analysis of Virulence Genes in Staphylococcus aureus Isolates

Among the total (30) virulence factors screened isolates, the percentage of sea was highest (93.75) %), followed by hla (87.5) %), hlb (75.0) %), tst (62.5%), and eta (53.36%). Among the identified virulence genes, 90% of them exhibited no difference in frequency between MRSA and macrolide isolates of clinical origin (P > .05), except for the eta gene, which had a higher percentage in macrolides than in MRSA isolates (P < .05). The sea percentage was higher in MRSA strains and in macrolide strains among food isolates (P < .05). The Hla, Hlb, eta, and tst carriage rates among isolates of clinical origin were significantly higher than those among isolates of vegetable origin (P < .05). In contrast, the sea carriage rates were higher among both vegetable and clinical isolates (P < .05), Figure 4.
Figure 4. PCR analysis of virulence factors in S. aureus: (eta 190bp, hla 209bp, hlb 309bp, tst 180bp, and sea 560bp) lanes 1-21 vegetable isolates and lanes 22-29 clinical isolates; lane 11 S. aureus ATCC29213 positive control

3.6. Analysis of the Macrolide and MRSA Resistance Gene Regions

In this study, four different multiplex PCR reaction mixtures were defined for four resistance genes to identify macrolide (erythromycin) and β-lactam (MRSA) resistant genes among the bacteria population. The antibiogram results showed 30 (96.8%), 29(93.5%), and 31 (100%) isolates were resistant to penicillin, macrolide (erythromycin) and methicillin, respectively. A total of four available resistant genes were analyzed in 10 antibiotic-resistant isolates using colony polymerase chain reaction (CPCR). There were 9(90%) positive results of macrolide-resistant ermA, 10(100%) positive results of ermB, 10(100%) positive results of ermC, 7(70%) positive results of msrA were determined as depicted in Figures 5 (A), (B), (C), and (D) and table 6 below.
Figure 5. A) PCR analysis of macrolide resistant determinants: erm(A) (139 bp), with vegetable & clinical isolates; source of DNA: 100bp DNA molecular size marker; S2: S3: S4; S6; S7: S8; C1: C2; C3: C4; C5 positive erm(A); B) PCR analysis of macrolide resistant determinants: msr(A) (163 bp) from vegetable & clinical isolates; source of DNA: 100 bp DNA molecular size marker; S4; S5: S6; S7: S8; C1: C2; C3 positive msr(A); C) PCR analysis of macrolide resistant determinants: erm B (142 bp), with vegetable & clinical strains; source of DNA: 100 bp DNA molecular size marker; S2: positive control; S3: S4; S 5: S6; S7: S8; C1: C2; C3 positive erm B gene; D) PCR analysis of macrolide resistant determinants: erm C (190 bp), with vegetable & clinical strains; source of DNA: 100 bp DNA molecular size marker; S2: positive control; S2, S3, S4, S5, S6, S7, S8, C1, C2, C3 positive erm C genes
Table 6. Distribution of Macrolide (erythromycin) and β lactamase (methicillin) resistance genes in Staphylococcus spp
     

4. Discussions

The primary focus of this study was to determine the total population and distribution of S. aureus in leafy vegetables and clinical sources from Gondar, Ethiopia including their antibiotic susceptibility, resistance genes, and other associated virulence factors. The results show that the vegetables had a high microbial load of Staphylococcus aureus [50].
The aerobic mesophilic count of leafy greens, the mean aerobic mesophilic count of cauliflower, lettuce, spinach, and cabbage were 7.9 cfu/g, 6.9 cfu/g, 6.7 cfu/g, and 6.4 cfu/g, respectively, while the ranges were, cauliflower (6.4-8.5 cfu/g, lettuce (5.5-8.4 cfu/g) spinach (5.5-7.4 cfu/g) and cabbage (5.3-7.5 cfu/g). The higher bacterial load in this study may be partly due to the use of contaminated irrigation water and organic fertilizers in the farms, coupled with the poor hygienic environment where open defecation is not uncommon. Staphylococcus aureus is one among the foremost common pathogenic bacteria within the clinic and within the community, and therefore the presence or absence of resistance and virulence genes is closely associated with the pathogenicity of S. aureus infection. Investigation of the mechanisms of antibiotic resistance and detecting the distribution of resistance and virulence genes in S. aureus isolates from different sources will guide rational drug use among medical personnel’s and stop the dissemination of infection [39].
The antibiogram of the S. aureus isolates revealed that, 99% of vegetable and clinical S. aureus isolates were multidrug resistant. At present, our data is above previous reports among various food resources in other countries [47,60,8,9]. Majority of the isolates (98.9%) were resistant to erythromycin, vancomycin, methicillin, and penicillin, showing the research of leafy vegetables (96.3%) in South Korea [25]. Resistance to other antibiotics including tetracycline (98%,98%), clindamycin (95%,95%), gentamicin (97%,97%), and ciprofloxacin (97%,97%) clinical and vegetable respectively, was also higher than the previous report, 49.4% of S. aureus isolates from food products were resistant to tetracycline, 24.1% to clindamycin, and 13.8% to gentamicin [14]. Considering these antibiotics are increasingly utilized in animal breeding or human treatment and exchange of antibiotic-resistant genes by mobile genetic elements (MGEs) [36], it is not surprising that resistant strains become more common within the present. As we all know, methicillin resistant S. aureus (MRSA) represents a significant public health issue due to its ability to colonize and infect humans and animals among the isolates of S. aureus during this study the antibiogram showed that each one was MRSA strains. Cross contamination from environments could also be the main reason because animal-derived food products are widely known to be a crucial reservoir for MRSA [60]. However, the high antimicrobial resistance of S. aureus observed during this study should receive much attention.
Macrolide and beta-lactam resistant gene regions (erm A, erm B, erm C, and misr A) were detected among the clinical and vegetable S. aureus isolates. Macrolide (erythromycin) resistance in staphylococci is encoded by erm gene [67]. All isolates found to be resistant to erythromycin by phenotypic methods contained a minimum of one erythromycin resistance gene. Our study confirmed that erm A, erm B, and msrA genes in S. aureus isolates were found at higher rates than in coagulase negative staphylococci, and among the coagulase negative staphylococci, ermC gene was more prevalent which agreed with previous reports [58]. In ten isolates, both erm(B) nor erm(C) gene was detected, although these were detected as resistant. Erythromycin resistance could be due to the presence of msrA or ermB genes as previously described in coagulase negative staphylococci [68]. Lim et al., reported that the ermA gene was more predominant than the opposite erythromycin resistance genes in S. aureus isolates, and the ermC gene was found mostly in coagulase negative staphylococci [69]. Similarly, during a study performed by Martineau et al., the ermC gene has been reported to be more prevalent in coagulase negative staphylococci. However, ermA has been reported to be the more common gene in coagulase negative in another study [70].
The isolates were also screened for virulence genes to work out their pathogenic risk. All clinical strains were positive for Hla, in accordance with a study conducted in Asturias, which found that each one strain isolated from cow milk harbored the Hla virulence gene [23]. Moreover, it had been reported that Hla was associated with an epidemic of staphylococcal gastrointestinal disorders [35]. Another important factor underlying staphylococcal gastrointestinal disorder is that the presence of enterotoxins, although the share of enterotoxin genes in isolates of food origin varies in several studies. Different levels of virulence genes indicate different pathogenic potentials [35]. This current study showed that sea was the highest (93.75) %), followed by hla (87.5 %), hlb (75.0) %), tst (62.5%), and eta (53.36%); these percentages were above those identified in chicken products from Egypt, [17] indicating that the genes may significantly contribute to staphylococcal gastrointestinal disorder.
The transmission of bacterial virulence factors to humans through the food chain as well as the higher multidrug resistance rates in those strains represents large risks for human infection. When resistance gene carriage rates were compared between MRSA and macrolide strains of food origin, only the etb rate was higher in MRSA strains; other genes demonstrated no differences. Combined with the results obtained for clinical isolates, our study suggested that the difference in virulence gene carriage rates between MRSA and macrolide strains might be insignificant [71].

5. Conclusions

In this study we have analyzed the microbial load of four different leafy greens in Gondar town at the point of retail sale. Our study takes a combined bioinformatical approach to acquire detailed data on antimicrobial resistant and virulence markers in S. aureus isolated from retail vegetables and clinical sources from Gondar community hospital. MRSA and Macrolide resistant isolates were detected among all the isolates. Resistance genes towards β-lactam antimicrobials, tetracycline, and erythromycin were frequently observed, which correlates with different antimicrobial classes being commonly used in Gondar clinical cares. In addition, common virulence gene were detected among the isolates. Generally, our results show that both clinical and vegetable isolates are resistant to all antibiotics. Due to the presence of antibiotic resistant S. aureus, as well as other opportunistic and emerging pathogens in the examined fresh vegetables and clinical sources, this study emphasizes the need for a new guideline to address this public health nuisance. In addition, as antibiotic resistance becomes a clear public health problem in the area, there needs to be closer monitoring by the health department.

ACKNOWLEDGEMENTS

I would like to express my sincere gratitude to Gondar University Department of Microbiology and Immunology and Howard University Biology department, applied microbiology laboratory.

Conflict of Interest

There is no conflict of interest with any party in this research work.

References

[1]  Amare, A., Worku, T., Ashagirie, B. et al. Bacteriological profile, antimicrobial susceptibility patterns of the isolates among street vended foods and hygienic practice of vendors in Gondar town, Northwest Ethiopia: a cross sectional study. BMC Microbiol 19, 120 (2019). https://doi.org/10.1186/s12866-019-1509-4.
[2]  Argudin MA, Tenhagen BA, Fetsch A, et al. Virulence and resistance determinants of German Staphylococcus aureus ST398 isolates from nonhuman sources. Appl Environ Microbiol. 2011; 77: 3052-3060.
[3]  Argudin MA, Tenhagen BA, Fetsch A, et al. Virulence and resistance determinants of German Staphylococcus aureus ST398 isolates from nonhuman sources. Appl Environ Microbiol. 2011; 77:3052-3060.
[4]  Ayçiçek et al., (2004). Assessment of the bacterial contamination on hands of hospital food handlers. Food Control. Volume 15, Issue 4, June 2004, Pages 253-259.
[5]  Becattini S. Doctoral dissertation, ETH Zurich. 2014.
[6]  Bindschedler, L. V., Panstruga, R., & Spanu, P. D. (2016). Mildew-Omics: How Global Analyses Aid the Understanding of Life and Evolution of Powdery Mildews. Frontiers in plant science, 7, 123. https://doi.org/10.3389/fpls.2016.00123.
[7]  Bougnom BP, Piddock LJ. ACS Publications. 2017.
[8]  Bryan Delaney, Richard E Goodman, Gregory S Ladics, Food and Feed Safety of Genetically Engineered Food Crops, Toxicological Sciences, Volume 162, Issue 2, April 2018, Pages 361–371, https://doi.org/10.1093/toxsci/kfx249.
[9]  Bryant, P.J., Huettner, B.H., Held, L.I., Ryerse, J., Szidonya, J., (1988). Mutations at the fat locus interfere with cell proliferation control and epithelial morphogenesis in Drosophila. Dev. Biol. 129: 541--554.
[10]  Clarridge J. E., 3rd (2004). Impact of 16S rRNA gene sequence analysis for identification of bacteria on clinical microbiology and infectious diseases. Clinical microbiology reviews, 17(4), 840–862. https://doi.org/10.1128/CMR.17.4.840-862.2004.
[11]  Clinical and Laboratory Standards Institute (2015) Performance Standards for Antimicrobial Susceptibility Testing; 25th Informational Supplement. CLSI Document M100-S25, Clinical and Laboratory Standards Institute, Wayne, PA.
[12]  Cuny C, Friedrich A, Kozytska S, Layer F, Nübel U, Ohlsen K, Strommenger B, Walther B, Wieler L, Witte W. Emergence of methicillin-resistant Staphylococcus aureus (MRSA) in different animal species. Int J Med Microbiol. 2010 Feb; 300(2-3): 109-17. doi: 10.1016/j.ijmm.2009.11.002. Epub 2009 Dec 16. PMID: 20005777.
[13]  David L. Smith, et.al., (2002). Animal antibiotic use has an early but important impact on the emergence of antibiotic resistance in human commensal bacteria. PNAS April 30, 2002 99 (9) 6434-6439.
[14]  Deyno, S., Fekadu, S., & Astatkie, A. (2017). Resistance of Staphylococcus aureus to antimicrobial agents in Ethiopia: a meta-analysis. Antimicrobial resistance and infection control, 6, 85. https://doi.org/10.1186/s13756-017-0243-7.
[15]  Doudoulakakis A, et al. Journal of Clinical Microbiology. 2017.
[16]  Duran N., et.al., (2012). Antibiotic resistance genes & susceptibility patterns in staphylococci. The Indian Journal of Medical Research. 2012 Mar; 135:389-396.
[17]  El Bayomi RM, Ahmed HA, Awadallah MA, et al. Occurrence, virulence factors, antimicrobial resistance, and genotyping of Staphylococcus aureus strains isolated from chicken products and humans. Vector Borne Zoonotic Dis. 2016; 16: 157-164.
[18]  Esfarjani, F., Khaksar, R., Mohammadi Nasrabadi, F., Roustaee, R., Alikhanian, H., Khalaji, N., Mousavi Khaneghah, A. and Hosseini, H. (2016), "A preventative approach to promote food safety: Bacterial contamination of domestic refrigerators", British Food Journal, Vol. 118 No. 8, pp. 2076-2091. https://doi.org/10.1108/BFJ-01-2016-0025.
[19]  Fair RJ, Tor Y. Perspectives in medicinal chemistry. 2014; 6: 25.
[20]  Feleke M. et.al., (2014). The growing challenges of antibacterial drug resistance in Ethiopia. Journal of Global Antimicrobial Resistance, Volume 2, Issue 3, September 2014, Pages 148-154.
[21]  Feleke, T., Eshetie, S., Dagnew, M. et al. (2018). Multidrug-resistant bacterial isolates from patients suspected of nosocomial infections at the University of Gondar Comprehensive Specialized Hospital, Northwest Ethiopia. BMC Res Notes 11, 602.
[22]  Fluit AC, Visser MR, Schmitz FJ (2001) Molecular detection of antimicrobial resistance. Clin Microbiol Rev 14:836–871.
[23]  Fueyo JM, Mendoza MC, Rodicio MR, et al. Cytotoxin and pyrogenic toxin superantigen gene profiles of Staphylococcus aureus associated with subclinical mastitis in dairy cows and relationships with macro-restriction genomic profiles. J Clin Microbiol. 2005; 43:1278-1284.
[24]  Gosden, P. E., Andrews, J. M., Bowker, K. E., Holt, H. A., MacGowan, A. P., Reeves, D. S. et al. (1998). Comparison of the modified Stokes's method of susceptibility testing with results obtained using MIC methods and British Society of Antimicrobial Chemotherapy breakpoints. Journal of Antimicrobial Chemotherapy 42, 161–9.
[25]  Hölzel, C. S., Tetens, J. L., & Schwaiger, K. (2018). Unraveling the Role of Vegetables in Spreading Antimicrobial-Resistant Bacteria: A Need for Quantitative Risk Assessment. Foodborne pathogens and disease, 15(11), 671–688.https://doi.org/10.1089/fpd.2018.2501.
[26]  Humphreys H, et al. Journal of Hospital Infection. 2016; 94:3.
[27]  Janda, J. M., & Abbott, S. L. (2007). 16S rRNA gene sequencing for bacterial identification in the diagnostic laboratory: pluses, perils, and pitfalls. Journal of clinical microbiology, 45(9), 2761–2764. https://doi.org/10.1128/JCM.01228-07.
[28]  Jarraud S. Relationships between Staphylococcus aureus genetic background, virulence factors, agr groups (alleles), and human disease. Infect Immun. 2002; 70:631-641.
[29]  Kesah C, Ben Redjeb S, Odugbemi TO, Boye CS, Dosso M, Ndinya Achola JO, Koulla-Shiro S, Benbachir M, Rahal K, Borg M. Prevalence of methicillin-resistant Staphylococcus aureus in eight African hospitals and Malta. Clin Microbiol Infect. 2003 Feb; 9(2): 153-6. doi: 10.1046/j.1469-0691.2003.00531. x. PMID: 12588338.
[30]  Kessie, G., M. Ettayebi, A.M. Haddad, A.M. Shibi, F.J. Al-Shammary A.F. Tawfik and M.N. Al-Ahdal, (1998). Plasmid profiles and antibiotic resistance in coagulase negative staphylococci isolated from polluted water. J. Applied. Microbiol., 84: pp.417-422.
[31]  Kumar, H.S., A. Parvathi and I. Karunasagar, (2005). Prevalence and antibiotic resistance of Escherichia coli in tropical seafood. World J. Microbiol. Biotechnol., 21: pp. 619-623.
[32]  Land M, Hauser L, Jun SR, Nookaew I, Leuze MR, Ahn TH, Karpinets T, Lund O, Kora G, Wassenaar T, Poudel S, Ussery DW. Insights from 20 years of bacterial genome sequencing. Funct Integr Genomics. 2015 Mar; 15(2): 141-61.
[33]  Landecker H. Body & Society. 2016; 22: 4.
[34]  Laxminarayan R, et al. The Lancet. 2016; 387.
[35]  Li G, Wu S, Luo W, et al. Staphylococcus aureus ST6-t701 isolates from food-poisoning outbreaks (2006-2013) in Xi’an, China. Foodborne Pathog Dis. 2015; 12:203‐206.
[36]  Lindsay JA. Staphylococcus aureus genomics and the impact of horizontal gene transfer. Int J Med Microbiol. 2014 Mar; 304(2): 103-9. doi: 10.1016/j.ijmm.2013.11.010. Epub 2013 Dec 1. PMID: 24439196.
[37]  Livermore DM. (2000). Antibiotic resistance in staphylococci. Int J Antimicrobial Agents. 16: S3–S10.
[38]  Luepke KH, et al. Pharmacotherapy the Journal of Human Pharmacology and Drug Therapy. 2017; 37:1.
[39]  Luo K, Shao F, Kamara KN, Chen S, Zhang R, Duan G, Yang H. Molecular characteristics of antimicrobial resistance and virulence determinants of Staphylococcus aureus isolates derived from clinical infection and food. J Clin Lab Anal. 2018 Sep; 32(7): e22456.
[40]  M. L. Delignette-Muller, J. P. Flandrois, (1994). An accurate diffusion method for determining bacterial sensitivity to antibiotics, Journal of Antimicrobial Chemotherapy, Volume 34, Issue 1, July 1994, Pages 73–81.
[41]  Mekonnen, S. A., et.al., (2018). Characterization of Staphylococcus aureus isolated from milk samples of dairy cows in small holder farms of North-Western Ethiopia. BMC veterinary research, 14(1), 246.
[42]  Mišić, M., Čukić, J., Vidanović, D., Šekler, M., Matić, S., Vukašinović, M., & Baskić, D. (2017). Prevalence of Genotypes That Determine Resistance of Staphylococci to Macrolides and Lincosamides in Serbia. Frontiers in public health, 5, 200.
[43]  Mritunjay, S. K., & Kumar, V. (2017). A study on prevalence of microbial contamination on the surface of raw salad vegetables. 3 Biotech, 7(1), 13.
[44]  Monteiro, A. S., Pinto, B., Monteiro, J. M., Ferreira, R. M., Ribeiro, P., Bando, S. Y., Marques, S. G., Silva, L., Neto, W., Ferreira, G. F., Biofilm, M., & Abreu, A. G. (2019). Phylogenetic and Molecular Profile of Staphylococcus aureus Isolated from Bloodstream Infections in Northeast Brazil. Microorganisms, 7(7), 210. Publisher Site | Google Scholar
[45]  OMEGA Innovations in nucleic acid isolation http://2015.igem.org/wiki/images/1/18/NUDT_CHIAN-Protoco1.pdf.
[46]  Otarigho, B., & Falade, M. O. (2018). Analysis of antibiotics resistant genes in different strains of Staphylococcus aureus. Bioinformation, 14(3), 113–122.
[47]  Papadopoulos P, Papadopoulos T, Angelidis AS, Kotzamanidis C, Zdragas A, Papa A, Filioussis G, Sergelidis D. Prevalence, antimicrobial susceptibility and characterization of Staphylococcus aureus and methicillin-resistant Staphylococcus aureus isolated from dairy industries in north-central and north-eastern Greece. Int J Food Microbiol. 2019 Feb 16; 291:35-41.
[48]  Qi Lin, Honghu Sun, Kai Yao, Jiong Cai, Yao Ren and Yuanlong Chi. The Prevalence, Antibiotic Resistance and Biofilm Formation of Staphylococcus aureus in Bulk Ready-To-Eat Foods. Biomolecules 2019, 9, 524.
[49]  Rodríguez-Lázaro, D., Oniciuc, E. A., García, P. G., Gallego, D., Fernández-Natal, I., Dominguez-Gil, M., Eiros-Bouza, J. M., Wagner, M., Nicolau, A. I., & Hernández, M. (2017). Detection and Characterization of Staphylococcus aureus and Methicillin-Resistant S. aureus in Foods Confiscated in EU Borders. Frontiers in microbiology, 8, 1344.
[50]  Santajit S, Indrawattana N. Mechanisms of Antimicrobial Resistance in ESKAPE Pathogens. Biomed Res Int. 2016; 2016:2475067. doi: 10.1155/2016/2475067. Epub 2016 May 5. PMID: 27274985; PMCID: PMC4871955.
[51]  Scarborough, P., et.al., (2014). Dietary greenhouse gas emissions of meat-eaters, fish-eaters, vegetarians, and vegans in the UK. Climatic Change 125 (2), 179-192.
[52]  Syne, S. M., et.al., (2013). Microbiological hazard analysis of ready-to-eat meats processed at a food plant in Trinidad, West Indies. Infection ecology & epidemiology, 3, 10.3402 /iee. v3i0.20450.
[53]  Thomer, L., Schneewind, O., & Missiakas, D. (2016). Pathogenesis of Staphylococcus aureus Bloodstream Infections. Annual review of pathology, 11, 343–364.
[54]  Udo E, Dashti A. Detection of genes encoding aminoglycoside-modifying enzymes in staphylococci by polymerase chain reaction and dot blot hybridization. Int J Antimicrob Agents. 2000; 13:273-279.
[55]  Van Tonder I., et.al., (2007). The personal and general hygiene practices of food handlers in the delicatessen sections of retail outlets in South Africa. J Environ Health. 2007; 70(4): 33-38.
[56]  Vanderhaeghen W, et al. Epidemiology & Infection. 2010; 138:5.
[57]  Wafaa Chaalal, Nadia Chaalal, Nadjette Bourafa, Mebrouk Kihal, Seydina M. Diene, and Jean-Marc Rolain. Characterization of Staphylococcus aureus Isolated from Food Products in Western Algeria, Jun 2018.
[58]  Weisblum B. Erythromycin resistance by ribosome modification. Antimicrob Agents Chemother. 1995 Mar; 39(3): 577-85. doi: 10.1128/aac.39.3.577. PMID: 7793855; PMCID: PMC162587.
[59]  World Health Organization (WHO), (2002). Global strategy for containment of antimicrobial resistant. WHO/CDS/CSRS/DRS/2001-2002.
[60]  Wu, S., Huang, J., Wu, Q., Zhang, F., Zhang, J., Lei, T., Chen, M., Ding, Y., & Xue, L. (2018). Prevalence and Characterization of Staphylococcus aureus isolated from retail vegetables in China. Frontiers in microbiology, 9, 1263.
[61]  Zapun A, Contreras-Martel C, Vernet T. Penicillin-binding proteins, and beta-lactam resistance. FEMS Microbiol Rev. 2008; 32:361–85.
[62]  Zhang KY, McClure JA, Elsayed S, et al. Novel multiplex PCR assay for characterization and concomitant subtyping of staphylococcal cassette chromosome mec types I to V in methicillin-resistant Staphylococcus aureus. J Clin Microbiol. 2005; 43: 5026-5033.
[63]  Zhang KY, McClure JA, Elsayed S, et al. Novel multiplex PCR assay for characterization and concomitant subtyping of staphylococcal cassette chromosome mec types I to V in methicillin-resistant Staphylococcus aureus. J Clin Microbiol. 2005; 43:5026-5033.
[64]  Zmantar, T., Kouidhi, B., Miladi, H., & Bakhrouf, A. (2011). Detection of macrolide and disinfectant resistance genes in clinical Staphylococcus aureus and coagulase-negative staphylococci. BMC research notes, 4, 453.
[65]  Janda, J. M., & Abbott, S. L. (2007). 16S rRNA gene sequencing for bacterial identification in the diagnostic laboratory: pluses, perils, and pitfalls. Journal of clinical microbiology, 45(9), 2761–2764. https://doi.org/10.1128/JCM.01228-07.
[66]  Spiliopoulou, I., Petinaki, E., Papandreou, P., & Dimitracopoulos, G. (2004). erm(C) is the predominant genetic determinant for the expression of resistance to macrolides among methicillin-resistant Staphylococcus aureus clinical isolates in Greece. The Journal of antimicrobial chemotherapy, 53(5), 814–817. https://doi.org/10.1093/jac/dkh197.
[67]  Ghanbari, F., Ghajavand, H., Havaei, R., Jami, M. S., Khademi, F., Heydari, L., Shahin, M., & Havaei, S. A. (2016). Distribution of erm genes among Staphylococcus aureus isolates with inducible resistance to clindamycin in Isfahan, Iran. Advanced biomedical research, 5, 62. https://doi.org/10.4103/2277-9175.179184.
[68]  Kitti, T., Seng, R., Saiprom, N., Thummeepak, R., Chantratita, N., Boonlao, C., & Sitthisak, S. (2018). Molecular Characteristics of Methicillin-Resistant Staphylococci Clinical Isolates from a Tertiary Hospital in Northern Thailand. The Canadian journal of infectious diseases & medical microbiology, Journal canadien des maladies infectieuses et de la microbiologie medicale, 2018, 8457012. https://doi.org/10.1155/2018/8457012.
[69]  Westh, H., Hougaard, D. M., Vuust, J., & Rosdahl, V. T. (1995). erm genes in erythromycin-resistant Staphylococcus aureus and coagulase-negative staphylococci. APMIS: acta pathologica, microbiologica, et immunologica Scandinavica, 103(3), 225–232.
[70]  Fair, R. J., & Tor, Y. (2014). Antibiotics and bacterial resistance in the 21st century. Perspectives in medicinal chemistry, 6, 25–64. https://doi.org/10.4137/PMC.S14459.
[71]  Chen, L., Yang, J., Yu, J., Yao, Z., Sun, L., Shen, Y., et al. (2005). VFDB: a reference database for bacterial virulence factors. J. Nucleic. Acids. 33, D325–D328.
[72]  Argudín, M. Á, Mendoza, M. C., and Rodicio, M. R. (2010). Food poisoning and Staphylococcus aureus enterotoxins. Toxins 2, 1751–1773. doi: 10.3390/toxins2071751.
[73]  Rinsky, J. L., Nadimpalli, M., Wing, S., Hall, D., Baron, D., Price, L. B., et al. (2013). Livestock-associated methicillin and multidrug resistant Staphylococcus aureus is present among industrial, not antibiotic-free livestock operation workers in North Carolina. PLoS One 8: e67641. doi: 10.1371/journal.pone.0067641.